Hisham M. Darwish

Diabetes mellitus: The epidemic of the century

The epidemic nature of diabetes mellitus in different regions is reviewed. The Middle East and North Africa region has the highest prevalence of diabetes in adults (10.9%) whereas, the Western Pacific region has the highest number of adults diagnosed with diabetes and has countries with the highest prevalence of diabetes (37.5%). Different classes of diabetes mellitus, type 1, type 2, gestational diabetes and other types of diabetes mellitus are compared in terms of diagnostic criteria, etiology and genetics. The molecular genetics of diabetes received extensive attention in recent years by many prominent investigators and research groups in the biomedical field. A large array of mutations and single nucleotide polymorphisms in genes that play a role in the various steps and pathways involved in glucose metabolism and the development, control and function of pancreatic cells at various levels are reviewed. The major advances in the molecular understanding of diabetes in relation to the different types of diabetes in comparison to the previous understanding in this field are briefly reviewed here. Despite the accumulation of extensive data at the molecular and cellular levels, the mechanism of diabetes development and complications are still not fully understood. Definitely, more extensive research is needed in this field that will eventually reflect on the ultimate objective to improve diagnoses, therapy and minimize the chance of chronic complications development.

Recent Advances in the Molecular Biology of Vitamin D Action

Following the cloning and deletion analysis of the vitamin D receptor, most recent advances have been in the isolation and characterization of the DNA response elements found in the promoter region of target genes of vitamin D. Vitamin D, like the thyroid and retinoid hormones, binds to repeat sequences, but the repeats are separated by three nonspecified bases. The action of the VDR requires the presence of the RXR proteins and evidently other proteins that are involved in regulating transcriptions. A possible role of phosphorylation of the ligand binding domain of the VDR in transcription has also appeared. Very likely, the molecular events involved in vitamin D stimulation or suppression of a target gene will include its interaction with a number of transcription factors, both in the regulation of transcription and in the actual machinery involved in the transcription process through polymerase II. Although likely, it is not entirely clear whether the genomic action of vitamin D can account for all of its biological activities. Nongenomic actions of the vitamin D hormone have been reported, but convincing evidence that this is of biological importance in vivo is lacking. Advances in our understanding of the vitamin D mechanism of action can clearly be expected from physical studies of cloned and expressed vitamin D receptor and its subdomains, elucidation of the transcription factors in vitamin D-modulated transcription of target genes, elucidation of the role of phosphorylation in the transcription process, and the identification of important genes that are regulated in the specific target tissues responsive to vitamin D. This will definitely remain as a very active field of investigation well into the future.

Phosphorylation is involved in transcriptional activation by the 1,25-dihydroxyvitamin D3 receptor

The 1,25-dihydroxyvitamin D3 receptor becomes phosphorylated upon treatment with 1,25-dihydroxyvitamin D3. We have investigated the role of phosphorylation in the transcriptional activity induced by 1,25-dihydroxyvitamin D3 through its receptor. An active 1,25-dihydroxyvitamin D3-dependent transcription system was reconstituted in CV-1 cells by co-transfection of plasmids containing the rat 1,25-(OH)2D3 receptor DNA and a functional vitamin D response element (DRE) in a reporter gene construct. Treatment of these transiently transfected CV-1 cells with modulators of protein kinase A (8-Br-cAMP, PKIA and H-9) and phosphatases (Okadaic acid) resulted in mimicking or abolishing the transcriptional activity of 1,25-dihydroxyvitamin D3 in a receptor-dependent fashion. These modulators directly altered 1,25-dihydroxyvitamin D3 receptor phosphorylation. Therefore, the present results strongly suggest that phosphorylation plays a central role in the transcriptional activity of the 1,25-dihydroxyvitamin D3 receptor.

20-Oxopregnacalciferols: Vitamin D compounds that bind the progesterone receptor

Abstract 20-Oxopregnacaliferols and 19-nor-20-oxopregnacalciferol were prepared from calciferol 22-aldehydes by an oxygenation procedure.

Transcriptional control of the osteocalcin gene by 1,25-dihydroxyvitamin D-2 and its 24-epimer in rat osteosarcoma cells

The effects of two vitamin D analogs, 1,25-dihydroxyvitamin D-2 and 24-epi-1,25-dihydroxyvitamin D-2, were examined on osteocalcin gene expression in the rat osteosarcoma cell line ROS 17/28. Our results indicate that these analogs are more transcriptionally active than 1,25-dihydroxyvitamin D-3, particularly the 24-epimer. Assessment of reporter gene chloramphenicol acetyltransferase (CAT) activity, using the vitamin D responsive element (VDRE) derived from the human osteocalcin gene promoter. revealed that both analogs stimulated CAT activity 5- to 10-fold. 1,25-Dihydroxyvitamin D-2 was slightly more active than 1,25-dihydroxyvitamin D-3, while the 24-epimer was twice as effective. 1,25-Dihydroxyvitamin D-3 also stimulated osteocalcin mRNA accumulation by 2-fold over vehicle-treated cells, 1,25-dihydroxyvitamin D-2 by 2.5-fold, and 24-epi-1,25-dihydroxyvitamin D-2 by 4-fold. Electrophoretic mobility shift assays using the osteocalcin vitamin D responsive element revealed no increase in DNA binding with either analog when compared to 1,25-(OH)2D3. Examination of CAT activity using the rat 24-hydroxylase VDRE indicated no significant difference in transcription with these compounds, suggesting that the vitamin D-2 analogs preferentially activate osteocalcin gene expression.

Association between factor V Leiden mutation and poor pregnancy outcomes among Palestinian women

Pregnancy is a hypercoagulable state with increased tendency for thrombus formation, a condition that is increased when combined with acquired or inherited risk factors that lead to thrombophilia. Among the inherited risk factors is Factor V Leiden mutation, an autosomal dominant trait with reduced penetrance. The mutation seems to be associated with different poor pregnancy outcomes including recurrent miscarriages. In the present study, we performed a case-control study to investigate the association between the Leiden mutation and poor pregnancy outcome among the Palestinian population in the West bank region of Palestine. The study included 145 subjects with recurrent miscarriages and 205 matched control subjects with successful pregnancies who experienced normal delivery and no apparent complications. Leiden mutation was detected in 41 of the 145 study subjects (28.2%), and in 24 of the 205 control subjects (11.7%). Subjects homozygous with the mutant allele were identified only among the test and not the control group. Data analysis indicates a significant association between the mutant allele and recurrent miscarriages (p-value<0.05). Furthermore, this association is significant between the mutant haplotype with miscarriages compared to control group showing time effect where there is no association for miscarriages before week 10. Results here also show strong association of factor V leiden polymorphism among primary aborters compared to secondary aborters or control groups. The odds ratio for the primary aborters was 75 and p<0.0001. In conclusion, these results provide evidence for a significant correlation between recurrent miscarriages and Factor V mutation in our population.

pectrum of β-Globin Gene Mutations Among Thalassemia Patients in the West Bank Region of Palestine

β-Thalassemia (thal) is an autosomal recessive disorder that results in hypochromic hemolytic anemia in affected patients. In the West Bank area of Palestine, the prevalence of β-thal trait is approximately 3.5% among the population, with an estimated 120,000 carriers. Seventeen β-globin gene mutations could be identified in 148 patients using polymerase chain reaction (PCR), amplification refractory mutation system (ARMS)-PCR and direct sequencing. The predominant mutations included: IVS-I-6 (T→C) (28.7%), IVS-I-110 (G→A) (17.6%), codon 37 (G→A) (10.4%), IVS-I-1 (G→A) (9%), codons 106/107 (+ G) (6.8%) and codon 39 (C→T) (4.6%). Other less frequent and rare mutations included: IVS-II-1 (G→A), codon 5 (–CT), IVS-II-848 (C→A), –30 (T→A), codons 8/9 (+ G), IVS-I-5 (G→C), –28 (A→C), IVS-II-745 (C→G), codon 6 (–A), codon 27 (G→T) and codon 30 (AGG→ACG). Most patients (62.2%) were homozygous for one type of mutation, while the rest (27.3%) were compound heterozygotes. Some patients were heterozygous for β-thal and sickle cell anemia traits. No mutations could be detected in both alleles of eight patients, while in seven patients only one mutant allele could be detected. Further investigations are needed to resolve the corresponding genotypes of these patients. This study represents a comprehensive investigation of the type, frequency, and distribution of thalassemia mutations among the Palestinian population in the West Bank region of Palestine. A degree of similarity and significant variations was evident in the type and frequency of mutations when the present mutations profile was compared with similar ones among various Arab and non Arab populations. The association between the identified mutations and the corresponding genotypes of our patients with specific polymorphism frameworks in the β-globin gene was performed and the results revealed linkage disequilibrium.

DNA bending is induced by binding of vitamin D receptor-retinoid X receptor heterodimers to vitamin D response elements

The ability of vitamin D receptor‐retinoid X receptor (VDR‐RXR) heterodimers to induce a DNA bend upon binding to various vitamin D response elements (VDRE) has been investigated by circular permutation and phasing analysis. Recombinant rat VDR expressed in the baculovirus system and purified recombinant human RXRβ have been used. The VDREs were from 1,25‐dihydroxyvitamin D3 (1,25‐[OH]2D3) enhanced genes (rat osteocalcin, rOC; mouse osteopontin, mOP, and rat 1,25‐dihydroxyvitamin D3‐24‐hydroxylase, r24‐OHase), and a 1,25‐(OH)2D3 repressed gene (human parathyroid hormone, hPTH). As shown by circular permutation analysis, VDR‐RXR induced a distortion in DNA fragments containing various VDREs. Calculated distortion angles were similar in magnitude (57°, 56°, 61°, and 59°, respectively for rOC, mOP, r24‐Ohase, and hPTH). The distortions took place with or without a 1,25‐(OH)2D3 ligand. The centers of the apparent bend were found in the vicinity of the midpoint of all VDREs, except for rOC VDRE which was found 4 bp upstream. Phasing analysis was performed with DNA fragments containing mOP VDRE and revealed that VDR‐RXR heterodimers induced a directed bend of 26°, not influenced by the presence of hormone. In this study we report that similar to other members of the steroid and thyroid nuclear receptor superfamily, VDR‐RXR heterodimers induce DNA bending. J. Cell. Biochem. 74:220–228, 1999.

Evaluation of Glycated Hemoglobin (HbA1c) for Diagnosing Type 2 Diabetes and Prediabetes among Palestinian Arab Population

The purpose of the study is to compare the potential of HbA1c to diagnose diabetes among Palestinian Arabs compared to fasting plasma glucose (FPG). A cross-sectional sample of 1370 Palestinian men (468) and women (902) without known diabetes and above the age of 30 years were recruited. Whole blood was used to estimate HbA1c and plasma for FPG and total lipid profile. Fasting plasma glucose was used as a reference to diagnose diabetes (≥ 126 mg/dL) and prediabetes (100–125 mg/dL). The area under the receiver operating characteristic curve (AUC) for HbA1c was 81.9% to diagnose diabetes and 63.9% for prediabetes. The agreement between HbA1c and diabetes as diagnosed by FPG was moderate (ĸ  =  0.498) and low with prediabetes (ĸ = 0.142). The optimal cut-off value for HbA1c to diagnose diabetes was ≥ 6.3% (45 mmol/mol). The sensitivity, specificity and the discriminant ability were 65.6% (53.1–76.3%), 94.5% (93.1–95.6%), 80.0% (72.8–87.3%), respectively. However, using cut-off value of ≥ 6.5% (48 mmol/mol) improved specificity. At this cut-off value, the sensitivity, specificity and the discriminant ability were 57.4% (44.9–69.0%), 97.1% (96.0–97.9%) and 77.3% (71.0–83.5%). For diagnosing prediabetes with HbA1c between 5.7–6.4% (39–46 mmol/mol), the sensitivity, specificity and the discriminant ability were 62.7% (57.1–67.9%), 56.3% (53.1–59.4%) and 59.5% (56.3–62.5%), respectively. HbA1c at cut-off value of ≥ 6.5% (48 mmol/mol) by itself diagnosed 5.3% and 48.3% as having diabetes and prediabetes compared to 4.5% and 24.2% using FPG, respectively. Mean HbA1c and FPG increase significantly with increasing body mass index. In conclusion, the ROC curves showed HbA1c could be used for diagnosing diabetes when compared to FPG but not for prediabetes in Palestinians Arabs even though only about 50% of the diabetic subjects were identified by the both HbA1c and FPG.

1,25-Dihydroxyvitamin D and not Calcium Is the Major Regulator of Calbindin-D 9-kDa mRNA Levels In Vivo

Abstract A possible role of calcium in vivo on intestinal calbindin-D 9-kDa mRNA levels has been studied in rats. In vitamin D-deficient rats, a marked increase in dietary calcium has a small but significant effect on calbindin-D 9-kDa mRNA levels, despite a dramatic increase in serum calcium concentration that clearly resulted from increased intestinal absorption of calcium. On the other hand, vitamin D under all circumstances increased calbindin-D 9-kDa mRNA levels, with the greatest levels found in animals on a low calcium diet where little or no calcium is available for absorption. These results strongly support the idea that 1,25-dihydroxyvitamin D is directly responsible for the induction of calbindin-D 9-kDa. [P.S.E.B.M. 1992, Vol 199]